The Effect Of PINK1/Parkin-Mediated Mitophagy On Aflatoxin B₁-Induced Immunosuppression In Mice Spleen

Aflatoxin B₁（AFB₁）can accumulate in spleen,causing spleen immunesuppression.Oxidative stress is an important pathological basis for the spleen immunesuppression caused by AFB₁,and can cause mitochondrial damage.Phosphatase and tensin homologues induce kinase-1（PINK1）/E3ubiquitin ligase（Parkin）mediated mitophagy can clear damaged mitochondria and maintain cell steady,but it is unclear whether it is involved in spleen immunesuppression caused by AFB₁.Therefore,taking Parkin as the point,proposes the hypothesis that"PINK1/Parkin-mediated mitophagy protects in the spleen immunesuppression induced by AFB₁",Firstly,60 wild-type（WT）C57BL/6 male mice were divided into a control group（CG）0 mg/kg AFB₁,low dose group（LG）0.5 mg/kg AFB₁,medium dose group（MG）0.75 mg/kg AFB₁,and high dose group（HG）1 mg/kg AFB₁,and treated 28 days;To detect the growth status,spleen immune function,oxidative stress,mitochondrial structure and function,and the expression of key factors in the PINK1/Parkin signaling pathway in mice,with the aim of revealing the correlation between mitophagy and spleen immunesuppression caused by AFB₁ and dose-response relationship,and screening the dose of AFB₁ used in Parkin intervention experiments;Subsequently,30 WT male C57BL/6 mice and 30Parkin knockout(Parkin^-/-)male C57BL/6 mice were randomly divided into WT control group（WCG）0 mg/kg AFB₁,WT AFB₁ group（WAG）1 mg/kg AFB₁,Parkin^-/-control group（PCG）0mg/kg AFB₁,and Parkin^-/-AFB₁ group（PAG）1 mg/kg AFB₁ and treated 28 days;The expression of key factors in the PINK1/Parkin signaling pathway,mouse status,and spleen immune function,oxidative stress,mitochondrial structure and function were detected.The aim of this study is to reveal whether PINK1/Parkin mediated mitophagy is involved in the spleen immunesuppression function induced by AFB₁ exposure,and its role.The test results are as follows:（1）AFB₁ toxicity test results1)Compared with CG,MG and HG mice body weight and spleen/body weight were significantly deduced,micrographic damaged,indicating that AFB₁ could induce the inhibite of growth and spleen damage of mouse.2)Compared with CG,MG and HG mice spleen gene expression of interleukin-2（IL-2）,IL-4,IL-6 and tumor necrosis factorα（TNF-α）was significantly decreased,indicating that AFB₁ could induce the suppression of spleen immune function in mice.3)Compared with CG,reactive oxygen species（ROS）and malondialdehyde（MDA）the contents of in the spleen of LG,MG and HG mice were significantly increased,and the activities of superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）were significantly decreased,indicating that AFB₁ could induce oxidative stress in mice spleen.4)Compared with CG,The mitochondria in the spleen tissue cells are damaged and mitophagythe levels of mitochondrial membrane potential（MMP）and adenosine triphosphate（ATP）content are significantly reduced in the spleen of MG and HG mice,indicating that AFB₁can induce the structural damage and functional inhibition of mitochondria in the spleen of mice.5)Compared with CG,the gene expression of PINK1,Parkin,ubiquitin adhesion regulator P62 protein（P62）and microtubule-associated protein l light chain 3（LC3）in MG and HG were significantly increased.Compared with CG,the expression of PINK1,phosphorylated Parkin（p-Parkin）,P62 and LC3II proteins in HG was significantly increased,indicating that AFB₁ could activate PINK1/Parkin-mediated mitophagy in mouse spleen.The above results showed that when mice were exposed to 1 mg/kg AFB₁,the spleen immune function was inhibited,and the mitophagy intensity was highest,so this dose was used as the dose for the subsequent Parkin gene intervention test.（2）The results of Parkin gene intervention test1)Compared with WAG,the expression of PINK1,P62 and LC3 gene in PAG decreased significantly,and the expression of PINK1,P62 and LC3II protein decreased significantly.It is suggested that Parkin^-/-can inhibit PINK1/Parkin-mediated mitophagy in mouse spleen.2)Compared with WAG,the body weight and spleen coefficient of PAG mice decreas ed significantly,and serious micrographic significantly damaged.This indicates that PINK1/Parkin-mediated mitophagy can alleviate the inhibition of spleen growth and structural dam age induced by AFB₁ in mice.3)Compared with WAG,the number of CD3⁺,CD4⁺and CD4⁺/CD8⁺T lymphocytes in the spleen of PAG mice was significantly decreased,the number of CD8⁺T lymphocytes was significantly increased,and the gene expression of IL-2,IL-4,IL-6 and TNF-αwere significantly enhanced.It is suggested that PINK1/Parkin-mediated mitophagy can alleviate the inhibition of spleen immune function induced by AFB₁.4)Compared with WAG,the contents of ROS and MDA in PAG mice spleen were significantly enhanced,the GSH-Px and SOD liveness in the spleen of PAG mice were remarkable diminished,indicating that PINK1/Parkin-mediated mitophagy could alleviate the oxidative stress of mice spleen induced by AFB₁.5)Compared with WAG,PAG mitochondrial ultrastructural damage increased,the level s of MMP and ATP content in PAG were significantly decreased.These results suggest tha t PINK1/Parkin-mediated mitophagy can alleviate AFB₁ induced mitochondrial damage.Conclusions:PINK1/Parkin mediated mitochondrial autophagy activation is involved in AFB₁ induced spleen immune suppression and plays a protective role,which is related to reducing oxidative stress and mitochondrial damage.